Urinary NGAL in gastrointestinal diseases can be used as an indicator of early infection in addition to acute kidney injury marker

Abstract Background and Aim Neutrophil gelatinase‐associated lipocalin (NGAL) is characterized by increased expression before the rise in serum creatinine and has been used as a biomarker for the early prediction of acute kidney injury (AKI). However, there have been no comprehensive analyses of its significance in gastrointestinal diseases. This study aimed to analyze the usefulness of measuring urinary NGAL levels in patients with gastrointestinal diseases. Methods This study included 171 patients with a wide range of gastrointestinal diseases. Urinary NGAL levels were measured in all patients within 24 h of admission and 72 h later. Results Urinary NGAL levels were higher in patients with acute pancreatitis and acute cholangitis/cholecystitis than in those with other diseases. Although lower than in these diseases, urinary NGAL tends to be higher in inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, as well as in acute and chronic liver diseases, and is higher in liver cirrhosis as the Child–Pugh grade increases. Furthermore, we found that the group with higher urinary NGAL levels, which continued to increase over time, had worse hospital stays and prognosis. Conclusion Urinary NGAL could be used as an indicator of infectious diseases rather than an indicator of AKI in inflammatory bowel diseases and cirrhosis, and could predict the prognosis of patients with gastrointestinal diseases.


Introduction
Neutrophil gelatinase-associated lipocalin (NGAL) is a 24 kDa glycoprotein belonging to the lipocalin superfamily that is released from activated neutrophils during infection and inflammation. 1NGAL was isolated and identified in 2002 as a protein that induced the differentiation of undifferentiated renal progenitor cells into epithelial cells and nephrons. 2][5] Although several clinical studies have reported the prognostic value of urinary NGAL, 6 it can be presumed that it can be elevated in many aspects of gastrointestinal diseases with infection and inflammation; however, there has been no comprehensive analysis of its overall significance.
This study aimed to capture the characteristics of the rise in urinary NGAL in each disease and find novels ways of utilizing urinary NGAL in patients with gastrointestinal diseases.We measured the levels in a wide range of gastrointestinal diseases, such as liver cirrhosis, gastrointestinal cancer, acute pancreatitis, acute cholangitis/cholecystitis, inflammatory bowel disease, and gastrointestinal bleeding.We first analyzed which aspects of these diseases are particularly prone to elevation.Additionally, further analysis of liver disease was conducted concerning liver function, inflammatory bowel diseases, and the prognostic significance of abnormal and unimproved NGAL levels in the field of gastroenterology, except for AKI.
University Medical and Dental Hospital between April 2020 and March 2022 for liver cirrhosis, acute hepatitis, cancer, acute pancreatitis, acute cholangitis/cholecystitis, inflammatory bowel disease (ulcerative colitis and Crohn's disease), and gastrointestinal bleeding.Patients undergoing maintenance hemodialysis were excluded from the study.Age, sex, BMI, presence of chronic kidney disease, intensive care unit (ICU) or high care unit (HCU) admission rate, length of hospital stay, and mortality were retrospectively examined.Serum and urine samples were collected from all patients within 24 h of admission and 72 h later to measure serum creatinine, eGFR, white blood cell (WBC) count, neutrophil count, neutrophil/lymphocyte rate (NLR), CRP, and urinary NGAL.Urinary NGAL levels were measured in an outsourced laboratory (BML, Japan).
We comprehensively examined the association between urinary NGAL levels and gastrointestinal diseases.In cirrhosis, we investigated the association between urinary NGAL levels and Child-Pugh grade. 7urther, we investigated the association between urinary NGAL levels and prognosis of gastrointestinal diseases.We defined the group that fulfilled both of the following criteria: the rising high group and the group that did not fulfill both criteria as the control group and analyzed the prognosis of patients in both groups.
AKI was defined by one of the following 8 ; 1.The serum creatinine level rises by more than 0.3 mg/dL within 48 h. 2. The serum creatinine level was known within the previous 7 days or increased by more than 1.5 times above the expected basal level.3. Urine volume decreased by 0.5 mL/kg/h over a 6-h period.
Chronic kidney disease was defined as one or both of the following conditions lasting for at least 3 months 9 : 1. eGFR<60 mL/min/1.73m 2 .2. Abnormal urinary findings such as proteinuria, a condition in which renal impairment is evident on imaging, blood tests, and pathological findings.
First, for all diseases, we analyzed the urinary NGAL levels on the first day of illness.The most prominent cases were acute pancreatitis (5 cases; 317.2 AE 254.5 ng/mL) and acute cholangitis/cholecystitis (21 cases; 367.5 AE 608.2 ng/mL) (Fig. 2).We analyzed the relationship between serum creatinine levels and the presence of AKI.Nineteen of the 26 patients (73.1%) had high urinary NGAL levels on the first sick day, 14 (53.8%) had high serum creatinine levels, and 11 (42.3%) had complications of AKI.In the present analysis, we found a correlation between urinary NGAL and serum creatinine (correlation coefficient: 0.701) (Fig. 3a) and considered that it could be an indicator of AKI, which has been conventionally described, but not necessarily for some diseases and could be used as another indicator.Therefore, further analysis was conducted.
Urinary NGAL is also relatively high in hepatic disorders and Crohn's disease.We focused on diseases in which urinary NGAL levels were above normal on the first day, except for acute pancreatitis and acute cholangitis/cholecystitis. Urinary NGAL was elevated in ulcerative colitis (39.7 AE 29.2 ng/mL) and Crohn's disease (71.9 AE 54.7 ng/mL), especially in higher cases of Crohn's disease.High urinary NGAL levels were also observed in cases of liver cirrhosis (39.4 AE 50.2 ng/mL) and acute hepatitis (66.0 AE 74.9 ng/mL).Urinary NGAL levels on the first day of illness were higher than the normal range in 80 of 171 patients (46.8%), including 7 of 13 patients (53.8%) with ulcerative colitis and 4 of 8 patients (50%) with Crohn's disease.However, there were no cases of ulcerative colitis complicated by AKI and only one case of Crohn's disease.Thus, in ulcerative colitis and Crohn's disease, NGAL was not necessarily associated with AKI, suggesting an increase due to intestinal infection (Fig. 3b).
Because of the variability in liver cirrhosis, we decided to conduct a more in-depth analysis of liver cirrhosis cases.
Figure 2 Urinary NGAL on the first sick day in all diseases.uNGAL on the first sick day was 39.7 AE 29.2 ng/mL for ulcerative colitis, 71.9 AE 54.7 ng/mL for Crohn's disease, 39.0 AE 56.0 ng/mL for gastrointestinal bleeding, 39.4 AE 50.2 ng/mL for liver cirrhosis, 66.0 AE 74.9 ng/mL for acute hepatitis, 317.2 AE 254.5 ng/mL for acute pancreatitis, 367.5 AE 608.2 ng/mL for acute cholangitis/cholecystitis, 46.3 AE 55.3 ng/mL for cancer, and 62.7 AE 51.6 ng/mL for others.Data are presented as the mean AE SEM.The orange bar is the reference value (≦30.5 ng/mL).uNGAL, urinary neutrophil gelatinase-associated lipocalin.
In liver cirrhosis, the trend is higher as the disease progresses-a finding that may suggest the presence of infection.Data from the first sick day of patients with liver cirrhosis were analyzed using the Child-Pugh classification system as follows: Grade A: 34 cases 25.0 AE 28.0 ng/mL; Grade B: 18 cases 48.2 AE 65.8 ng/mL; Grade C: 9 cases 70.2 AE 56.7 ng/mL; as grade increased, urinary NGAL was noticeably higher (Fig. 4a).Urinary NGAL on the first sick day was high in 4 of 34 patients (11.8%) in Grade A, 10 of 18 patients (55.6%) in Grade B, and 6 of 9 patients (66.7%) in Grade C. Serum creatinine in the first sick day was Grade A: 34 cases 0.91 AE 0.19 mg/dL, Grade B: 18 cases 0.90 AE 0.73 mg/ dL, Grade C: 9 cases 2.1 AE 1.5 mg/dL, showing no significant difference between Grade A and Grade B (Fig. 4b).In Grade A, there were no cases of AKI; however, four of nine cases (44.4%) of Grade C were associated with AKI.On the other hand, CRP was Grade A: 34 cases 0.63 AE 1.2 mg/dL; Grade B: 18 cases 0.77 AE 1.7 mg/dL; Grade C: 9 cases 4.4 AE 4.1 mg/dL, CRP tended to increase as the grade increased (Fig. 4c).Although urinary NGAL levels tended to increase as the Child-Pugh grade increased, there were many cases with abnormal values that were not necessarily associated with AKI (Fig. 3c).
This suggests that as liver cirrhosis progresses, there is inflammation in the body that gradually activates neutrophils and is associated with AKI, especially in Child-Pugh Grade C.However, in Grade B, urinary NGAL levels are high, but Patients with urinary NGAL > 30.5 ng/mL on Day 1 and urinary NGAL on Day 3 > urinary NGAL on Day 1 tended to have a worse prognosis.We investigated whether urinary NGAL level could be a prognostic indicator of gastrointestinal diseases.We noticed that not only abnormal urinary NGAL on Day 1 but also patients who did not show improvement in urinary NGAL levels from Day 1 to Day 3 had a poor prognosis.

Discussion
In this study, we analyzed the usefulness of urinary NGAL levels in a wide range of gastrointestinal diseases.Urinary NGAL levels tend to be higher in patients with acute pancreatitis and acute cholangitis/cholecystitis than in those with other diseases.Although lower than in these diseases, urinary NGAL tends to be higher in inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, as well as in acute and chronic liver diseases, and is higher in liver cirrhosis as the Child-Pugh grade increases.We considered that these could be used as indicators of infectious diseases rather than necessarily as indicators of AKI.We found that the group with higher urinary NGAL levels, which continued to increase over time, had worse hospital stay and prognosis.
Most studies on NGAL have focused on its relationship with AKI, a clinical syndrome that causes a sudden loss of renal function and renal tissue damage.Indeed, Mishra et al. reported the usefulness of NGAL as a marker for the early diagnosis of AKI after cardiac surgery. 10In gastrointestinal diseases, Pradeep Siddappa et al. reported the usefulness of NGAL as a marker to predict the severity of acute pancreatitis and AKI. 1 AKI is often present in many severe phases of the disease and is often directly related to prognosis, so it is not surprising that NGAL is an important indicator.However, because NGAL is first released by activated neutrophils, 11 we believe that it should be more closely examined as a marker reflecting the activated state of neutrophils and not be limited to AKI.In addition, NGAL is significantly elevated in acute pancreatitis and cholangitis without AKI.Min et al. reported the usefulness of NGAL as a marker to predict ICU admission rates and mortality due to pneumonia. 12n support of our data, Allegretti et al. reported the usefulness of NGAL as a prognostic marker for patients with end-stage liver cirrhosis. 6Further, Gambino et al. reported that urinary NGAL is an independent predictor of in-hospital mortality. 13We are particularly interested in the relationship between increased portal pressure and NGAL associated with progressive fibrosis in liver cirrhosis.In liver cirrhosis, severe dysbiosis is caused by various factors, including portal hypertension, submucosal edema, difficulty for immune cells to infiltrate, abnormal interepithelial tight junction (TJ) protein, disruption of the gut vascular barrier (GVB), and decreased farnesoid X receptor (FXR) signaling in the ileum, which also results in decreased barrier function and abnormal bacterial products and metabolites. 14,15In patients with liver cirrhosis, the invasion of enteric bacteria and bacteria-associated substances, such as lipopolysaccharide (LPS) and pathogen-associated molecular patterns (PAMPs) (bacterial invasion is called bacterial translocation), can cause extremely serious liver and systemic conditions. 16These not only cause liver damage, but also lead to systemic inflammation.These conditions lead to cirrhosis-associated immune dysfunction (CAID).The CAID is divided into two categories: low and high grade.It can be divided into low-grade systemic inflammation, which is observed in patients with compensated cirrhosis without organ failure and some decompensated cirrhosis, and high-grade systemic inflammation, which is observed in patients with acuteon-chronic liver failure (ACLF) and is associated with high rates of short-term mortality.8][19] We presume that NGAL may capture latent infection and inflammation due to the invasion of such bacteria and their components, and AKI, as represented by hepatorenal syndrome, is a related symptom, although it may suggest prerequisite infection and inflammation.Similar to leaky gut in cirrhosis, many cases of inflammatory bowel disease causing intestinal barrier dysfunction also show elevated NGAL levels without renal dysfunction.Interestingly, Abdelsameea et al. showed that not only does urinary NGAL tend to be higher in cirrhosis, but it is also significantly higher in hepatocellular carcinoma.Although we did not search for hepatocellular carcinoma cases in this study, this is a very interesting report that also explores the mechanism behind the elevated levels. 20Oikonomou et al. reported that NGAL is elevated in active IBD and correlates with established inflammatory markers and disease activity.This finding strongly suggests that this marker may capture latent infection and inflammation in these diseases. 21ur study has some limitations.The patients receiving drugs that cause tubular necrosis, such as nonsteroidal antiinflammatory drugs (NSAIDs), were not excluded from this study.Also, this study shows that the number of cases per disease was reduced due to the analysis of a wide range of diseases.Despite these limitations, we believe that this study is significant because it identifies a new way to utilize NGAL in gastrointestinal diseases.
Despite these limitations, we have found new implications for urinary NGAL measurement in gastrointestinal diseases.Urinary NGAL may be used not only as an indicator of AKI but also as an indicator of infection in some diseases such as inflammatory bowel disease and liver cirrhosis.Furthermore, it has been suggested to be prognostic in a wide range of gastrointestinal diseases.

Figure 3
Figure 3 Correlation between urinary NGAL and serum creatinine.(a) Correlation between urinary NGAL and serum creatinine in acute pancreatitis and acute cholangitis/cholecystitis. (b) Correlation between urinary NGAL and serum creatinine in ulcerative colitis and Crohn's disease.(c) Correlation between urinary NGAL and serum creatinine in liver cirrhosis.sCre, serum creatinine; uNGAL, urinary neutrophil gelatinase-associated lipocalin.

Figure 4
Figure 4 Comparison of urinary NGAL, serum creatinine, and CRP by Child-Pugh classification in liver cirrhosis.(a) Comparison of uNGAL by Child-Pugh classification.(b) Comparison of sCre by Child-Pugh classification.(c) Comparison of CRP by Child-Pugh classification.The orange bar is the reference value (≦30.5 ng/mL).CRP, C-reactive protein; sCre, serum creatinine; uNGAL, urinary neutrophil gelatinase-associated lipocalin.Mann-Whitney U analysis.

Table 1
Diseases of patients

Table 3
Univariate and multivariate analyses of prognostic indicators of gastrointestinal disease using urinary NGAL Satisfy ① and ② (n = 18) Not satisfy ① and ② (n = 153) Day 1 NGAL > 30.5 ng/mL.②Urine Day 3 NGAL > Urine Day 1 NGAL.P < 0.05 was defined as a significant difference.Y Kojima et al.Urinary NGAL as a marker of infection